Measurement of cholesterol levels in plasma that are associated specifically with high-density lipoprotein (HDL) particles is of great interest in view of the nexus between cholesterol levels and cardiovascular problems. Current methods are typically based on enzymatic reactions. If total cholesterol (TC) is to be measured, which is a combination of free cholesterol (FC) and cholesterol ester (CE), three enzymes are typically used—cholesterol esterase, also known as cholesterol ester hydrolase (CEH), cholesterol oxidase and peroxidase along with reagents for detecting hydrogen peroxide.
When free cholesterol is measured in HDL or plasma, only two of these enzymes are used—cholesterol oxidase and peroxidase. Monitoring a change in free cholesterol concentration is routinely used to measure LCAT activity, which catalyzes conversion of free cholesterol to cholesteryl esters with acyl groups from phosphatidylcholine. Inhibitors of this enzyme, such as iodoacetate are often added to the LCAT assay reaction mixture to act as a negative control. Unfortunately, this also alters the activity of the enzymes cholesterol oxidase and/or peroxidase, critical to the free cholesterol assay.
Thus, for example, in a typical result using a commercially available enzyme-based assay employing cholesterol ester hydrolase, cholesterol oxidase and peroxidase and reagents to develop color, increasing the iodoacetate concentration from 0-250 mM lowers the estimated cholesterol content of a test solution containing 200 mg/dL of free cholesterol from 200 mg/dL when no iodoacetate is added to 175 mg/dL when 200 mM of iodoacetate is included in the reaction mixture. Clearly it would be advantageous to eliminate these artifacts.
It is known in the art that macrolide antibiotics—i.e., polyene, polyol macromolecules are able to complex with cholesterol to result in complexes that can be determined using optical spectroscopy by virtue of changes in their fluorescence spectra or optical density spectra. The present invention takes advantage of this complexation to assess the level of cholesterol in blood or plasma or fractions thereof, or in test samples generally.